Apparatus and methods for high-resolution and high-sensitivity assays

ABSTRACT

An apparatus allows separation, fractionation isolation and fraction collection simultaneously. The device consists of two major pieces, with one piece slides relatively to the other to facilitate the switching between separation and fractionation.

This is a continuation of U.S. application Ser. No. 10/371,981 filed onFeb. 21, 2003, now U.S. Pat. No. 7,189,370 which claims the priority ofthe following pending applications: U.S. Provisional Patent ApplicationSer. No. 60/359,391, filed on Feb. 22, 2002; U.S. Provisional PatentApplication Ser. No. 60/383,190 filed on May 3, 2002; and U.S. Utilitypatent application Ser. No. 10/076,012 filed on Feb. 11, 2002.

FIELD OF THE INVENTION

The present invention relates generally to the field of sensitivedetection of molecules. More particularly, the present invention relatesto methods and apparatus of use in multi separation techniques tosensitively measure the quantities of extracts of herbs, plants,organisms/tissues/biological fluids and other natural materials, andprotein/peptide samples, using a novel apparatus that integrates oneanalytical technique to another analytical technique.

DESCRIPTION OF RELATED ARTS

The technology advancement has greatly facilitated the separation andidentification of proteins in complex protein mixtures. The most populartechnologies include the two-dimensional gel electrophoresis (2DE)followed by mass spectrometry (MS) detection, the difference gelelectrophoresis (DIGE), the shotgun approach, the isotope-coded affinitytag (ICAT) technique, etc. A common argument in favor of 2DE is that acomparison can readily be made between two gels and thus proteomedifferences can be detected. However, 2DE is still problematic becauseof the gel-to-gel irreproducibility, varying staining efficiency ofindividual gels, and bias against some protein classes such as membraneproteins. Due to the huge differences in the distribution of proteins incomplex proteomes of humans, the detection and identification ofproteins expressed in low copy numbers is a major challenge. The lowabundance of important physiologically relevant proteins has renderedtheir analyses almost impossible without some means of priorpurification and enrichment from tissue lysates or biological fluids.All these problems are more or less resulted from the inadequate LOD anddynamic range of the analytical tools that are being used. Consideringthe frequency at which post-translational modifications of proteinsoccur, the separation of protein isoforms is essential to understandingbiological changes, and 2DE remains to be one of the main techniquesthat can offer sufficient resolution to address this issue at afunctional level. In this project, we will push the limits (e.g. LOD anddynamic range) of 2DE for LAP profiling. The following section presentsan overview of 2DE and the related technologies.

2DE. The first 2-dimensional (2D) separations can be attributed to thework of Smithies and Paulik who used a combination of paper and starchgel electrophoresis for the separation of serum proteins. Subsequentdevelopments in electrophoretic technology, such as the use ofpolyacrylamide as a support medium and the use of polyacrylamideconcentration gradients, were rapidly applied to 2D separations. Inparticular, the application of isoelectric focusing (IEF) techniquesdeveloped by O'Farrell to 2D separations made it possible for the1^(st)-D separation to be based on the charge properties of theproteins. The coupling of IEF with SDS-PAGE in the 2^(nd)-D resulted ina 2DE method that separated proteins according to two independentparameters, isoelectric point and molecular weight. This methodology wasthen adapted to a wide range of samples with differing solubilityproperties by the use of urea and applied to the analysis of proteinmixtures of whole cells and tissues.

There are three basic methods for the 1^(st)-D, ISO-Dalt, IPG-Dalt, andNEpHGE. In ISO-Dalt, the pH gradient is formed by carrier ampholytesduring the separation. At equilibrium, each protein is focused to aposition corresponding to its isoelectric point. The disadvantage ofthis technique is the lot-to-lot variability of the carrier ampholytes.ISO-Dalt is still used because the gels can be prepared easily withouthaving to make sophisticated gradients. IPG-Dalt uses an immobilizedampholyte strip to create a pH gradient. This technique provides betterbatch-to-batch reproducibility because the strips are commerciallyavailable in a dry state that can be easily hydrated. However, problemshave been reported, such as loss of large proteins in the 1^(st)-D,unsatisfactory resolubilization in the 2^(nd)-D, and precipitation byunpolymerized immobilines. Non-equilibrium pH gradient electrophoresis(NEpHGE) is typically used to resolve extremely basic proteins. Itdiffers from the other two in that: (i) the polarity of the power supplyis reversed at a predetermined volt-hour value during the separation;and (ii) the focusing process is stopped before reaching equilibrium.

After the 1^(st)-D separation, the buffer in the gel is exchanged withSDS buffer. The partially resolved proteins from the 1^(st)-D are thentransferred to a slab-gel for SDS-PAGE, the 2^(nd)-D separation. At theend of the 2^(nd)-D separation, the separated proteins are stained anddetected. The LOD and dynamic range depend on the stain method that isused.

Common stain methods. Coomassie Brilliant Blue is often utilized tostain proteins in SDS-PAGE gels for both qualitative and quantitativedetections. The advantages of Coomassie staining methods includequantitative binding of dye to proteins, low price, and goodreproducibility. Usually, it is compatible with MS analysis. When aprotein is detectable with Coomassie Brilliant Blue, as a rule of thump,enough protein is present for appropriate mass spectrometry analysiswith MALDI-TOF. The disadvantages are the long staining times,relatively low sensitivity, and narrow dynamic range.

Silver Staining is another popular staining method employed for proteindetection. In this method, the gel is preserved with soluble silver ionsand developed by treatment with formaldehyde, which reduces silver ionsto form an insoluble brown precipitate of metallic silver. Thisreduction is promoted by protein. It is a sensitive (sub-nanogramdetection limit) method for permanent staining of proteins in SDS-PAGEgels. However, it is incompatible with MS, and has a narrow dynamicrange.

Fluorescent dye staining is another technology that is widely used forproteome analysis due to its advantages of high sensitivity combinedwith wide linear dynamic range. For example, SYPRO Ruby Staining has asensitivity of ˜1 ng per spot and a linear dynamic range of ˜10³. Thesenumbers reflect the highest level of state-of-the-art technology, buthigher sensitivities and wider dynamic ranges are still greatlydemanded.

Electroblotting of proteins from 2DE. Because of the ability of 2DE toseparate simultaneously up to several thousand proteins usinglarge-format gels, it has become the method of choice for the analysisof protein expression in complex biological systems. A variety ofmethods are available now to further identify and characterize proteinsseparated by 2DE. Many of these methods depend on the technique ofWestern electroblotting in which proteins separated by 2DE aretransferred (“blotted”) by the application of an electric fieldperpendicular to the plane of the gel onto the surface of an inertmembrane, such as nitrocellulose.

Two types of apparatus are in routine use for electroblotting. In thefirst approach (known as “tank” blotting), a sandwich assembly of geland blotting membrane is placed vertically between two platinum wireelectrodes arrays contained in a tank filled with a blotting buffer.¹⁹In the second type of procedure (know as “semidry” blotting), thegel-blotting membrane assembly is sandwiched between two horizontalplate electrodes, typically made of graphite.

Proteins immobilized in this way are readily accessible to interactionwith probes, such as polyclonal antibodies and monoclonal antibodies orother ligands specific for the proteins being analyzed. This approachhas been used extensively for specific (known) protein detection andquantitation. Recently, 2DE is used for micro-preparative purificationof proteins for subsequent chemical characterization, which has oftenbeen applied for proteomic research.²¹ In this approach the protein,while still on the surface of the inert membrane support, can beanalyzed by numerous characterization techniques, including N-terminaland internal amino acid sequencing, amino acid compositional analysis,peptide profiling, and mass spectrometry.

Capillary SDS-PAGE. Capillary SDS-PAGE is a miniaturized gelelectrophoresis platform that has many advantages over traditionalslab-gel techniques (e.g. higher separation efficiency, shorterseparation time, lower mass detection limit, more convenient toimplement automated operation, etc.). Capillary SDS-PAGE is a specialtype of CGE, but for the simplicity of expression in the text of thisproposal we treat it as CGE. Typically, the separated proteins in CGEare detected in-column by either an Ultraviolet (UV) absorbance or alaser-induced fluorescence (LIF).

UV absorption is arguably the most frequently used detection mode in CE.It is also commonly employed in CGE since protein-SDS complexes absorbslight around 280 nm due to the aromatic side groups of amino acids andaround 200˜220 nm due to the peptide bonds between amino acids. LIFdetection is preferred when a low limit of detection and a wide dynamicrange are desired. Native fluorescence of proteins has been explored fordirect protein detection, but it is not widely accepted because of theuse of expensive UV lasers. Often, proteins are somehow fluorescentlylabeled, and then measured by a LIF detection system using a relativelyinexpensive laser such as an air-cooled argon ion or helium-neon laser.

Labeling proteins reliably and reproducibly is challenging, althoughmuch progress has been made. Proteins have been covalently linked withfluorescent dyes mainly via the amine groups on the proteins. Theseproteins can then be bound with SDS and separated by CGE. Proteinconcentrations of as low as 3×10⁻¹⁰ M (4˜10 ng/mL) were successfullyanalyzed by CGE. However, most of these protocols are cumbersome andsuffer from incomplete and ambiguous labeling, resulting in complexelectropherograms. Alternatively, proteins can react with SDS first andthe protein-SDS complexes are then dynamically labeled with fluorescentdyes before electrophoresis. Presumably due to the low bindingefficiency and high background noise, only moderate low LODs (30˜500ng/mL) were achieved.

This invention describes a method and the associated instrument to mapthe LAPs in complex biological samples. To reduce the HAP interferencewith the detection of the LAPs, we deplete the HAPs from the biofluidsusing affinity depletion techniques. The IEF fractionation device canseparate/concentrate/fractionate the LAPs, and all fractions can befurther separated by parallel CGE. The method and apparatus can achievea LOD of ≦10×10⁻¹⁵ mol/mL, a dynamic range of ≧10⁵ and a resolving powerof ≧5,000 proteins for LAP mapping and profiling.

SUMMARY OF THE INVENTION

The present invention solves a long-standing need in the art byproviding a high-resolution fractionation device for high resolution andhigh sensitivity assays of protein and peptide samples. The apparatuscan be used for variety of applications, including extracts of herbs,plants, organisms/tissues/biofluids and other natural materials,microorganisms, DNA, RNA, carbohydrates, polysaccharides and lipids.

In one aspect of the present invention, the fractionation deviceincorporates a separation scheme, such as IEF. The pre-separatedfractions are isolated locally to maintain the resolution of the abovesaid separation.

In one embodiment, a pre-separated sample (e.g. from an HPLC or a CEinstrument) is first introduced into the fractionation device. Thefractionation device then segments and isolates the said sample locallybefore fractionation.

In one particular embodiment, the integrated device has 2 pieces. Thetop piece moves relatively to the bottom piece to facilitate thepre-separated sample bands to be transferred to fraction collectioncontainers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the fractionation device

FIG. 2. Schematic design of the fractionation device assembly

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a fractionation device for highresolution and high sensitivity assays of complex biological samples,such as herb extracts and protein/peptides samples from proteomic field.FIG. 1 presents a schematic diagram of the fractionation device. Becauseit operates like a valve, it is sometimes called a valve device. Thedevice consists of two pieces (X and Y) and works as a sliding valve.The thin and thick lines represent capillary tubes. Pieces X and Y canbe made of materials such as PEEK (polyetherether ketone), nylon, PVC,alumina, ceramic and silica. Holes will be drilled on X and Y strips tohost the capillaries. Adhesives can be applied to secure the capillariesin position. When pieces X and Y are aligned as depicted in FIG. 1A, thecapillaries indicated by the thin lines are connected, forming acontinuous tube. Separations such as isoelectric focusing (IEF) andisotachophoresis can be performed in this tube. After IEF, piece Xslides to the other position as depicted in FIG. 1B to segment andisolate the pre-separated compounds. The segmented samples are thencollected into different vials (without loss of any sample) for the2^(nd)-D separation. Note: the pre-separated samples are collected onboth piece X and piece Y.

FIG. 2A presents a schematic arrangement of the valve device assembly toenable piece X to switch back and forth as illustrated in FIG. 1. PiecesX and Y are held together by two blocks using bolts and nuts via fourthrough holes (see FIG. 2C for the positions of the four holes). FIG. 2Bexhibits a cross-section view and FIG. 2C displays the top-view of theblock holding piece X. Referring to FIG. 2B, a pocket is machined on thebottom of the block. The pocket has a depth that is slightly (e.g. 1 mm)shallower than the thickness of piece X. This allows piece X to extrudeout slightly to ensure good sealing between pieces X and Y when theassembly is tightened (see the interface between pieces X and Y in FIG.2A). The length of the pocket equals to the length of piece X plus thesliding distance, which allows the ends of the pocket to serve as twostoppers for the valve switching. An open slit will be made in themiddle of the Nylon block (see FIG. 2C) to permit the attachedcapillaries coming out. Two holes are drilled perpendicular to the openslit so that two pins can be inserted in to secure the two switchingbars. The bottom Nylon block is used to hold piece Y in a fixedposition. Referring back to FIG. 2A, by pushing the right switching barto the right, piece X will move to the left and stop as it hits theleft-end of the pocket. By pushing the left switching bar to the left,piece X will move to the right and stop as it hits the right-end of thepocket.

The following presents an example protocol to perform an IEFseparation/fractionation.

-   -   1. Prepare the IEF sample by mixing ampholytes with a protein        sample    -   2. Set the fractionation device to the position as depicted in        FIG. 1A    -   3. Load the sample into the continuous capillary (the thin-line        of FIG. 1A)    -   4. Run IEF    -   5. Turn off the HV for IEF and switch the device to the other        position as depicted in FIG. 1B    -   6. Collect the proteins inside the segmented capillaries        (Although we have “split” the sample into 100 fractions,        proteins of similar pI should be “focused” in one or two        fractions after IEF. That is, the resolution of IEF is        retained.)

All of the methods and apparatus disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the invention has been described with respect to thedescribed embodiments in accordance therewith, it will be apparent tothose skilled in the art that various modifications and improvements maybe made without departing from the scope and spirit of the invention.For example, it will be apparent to those of skill in the art thatvariations may be applied to the methods and apparatus and in the stepsor in the sequence of steps of the methods described herein withoutdeparting from the concept, spirit and scope of the invention. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims. Accordingly, it is to beunderstood that the invention is not to be limited by the specificillustrated embodiments, but only by the scope of the appended claims.

1. A device for high-resolution and high sensitivity assays, comprising:at least a first block and a second block that are configured to sliderelative to each other from a first position to a second position; atleast two tubes that are attached to at least one of the said blocks; atleast a semi-looped channel segment that that is attached to or embeddedin at least one of the said blocks; wherein at the first position, thedevice is configured so that at least two of the said semi-loopedchannel segments are joined together forming a single continuouschannel, and wherein at the second position, the device is configured sothat the said continuous channel is broken into at least two channelsegments, at least one of the segments is in fluidic connection with twotubes that are attached to one of the said blocks;